Transcriptional analysis of human survivin gene expression.
Transcriptional analysis of human survivin gene expression.
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DOI：
10.1042/bj3440305
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发表时间：
1999-12
期刊：
影响因子：
--
通讯作者：
Fengzhi Li;D. Altieri
中科院分区：
文献类型：
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作者：
Fengzhi Li;D. Altieri
组织和器官的稳态的维持依赖于控制细胞凋亡(程序性细胞死亡)的基因的调控表达。在这项研究中，我们研究了Survivin基因的基本转录要求，Survivin基因是一种在癌症中显著上调的IAP(凋亡抑制物)。对人类Survivin基因5‘侧翼区的分析表明，存在一个非TATA启动子，该启动子含有一个约250个核苷酸的典型CpG岛、三个细胞周期依赖元件、一个细胞周期同源区和大量的Sp1位点。用甲基化敏感和不敏感的限制性内切酶对人类基因组DNA进行的聚合酶链式反应分析表明，CpG岛在正常组织和肿瘤组织中都没有甲基化。人Survivin基因的引物延伸和S1核酸酶图谱表明，在起始ATG的-72位和-57/-61位之间有两个主要的转录起始点。用截短的或嵌套的Survivin启动子-荧光素酶载体转染宫颈癌HeLa细胞，发现同时存在增强子和抑制子序列，并在人Survivin基因-230nT的近端发现了一个最小的启动子区域。对人Survivin启动子的无偏向突变分析表明，将Sp1序列定位在-171和-151位可使基础转录活性降低约63-82%。DNA寡核苷酸的电泳迁移率改变分析证实，Survivin Sp1序列与HeLa细胞提取物之间形成了DNA-蛋白质复合体，该反应被Survivin Sp1位点的突变所消除。这些发现确定了Survivin基因表达的基本转录要求。
The preservation of tissue and organ homoeostasis depends on the regulated expression of genes controlling apoptosis (programmed cell death). In this study, we have investigated the basal transcriptional requirements of the survivin gene, an IAP (inhibitor of apoptosis) prominently up-regulated in cancer. Analysis of the 5' flanking region of the human survivin gene revealed the presence of a TATA-less promoter containing a canonical CpG island of approximately 250 nt, three cell cycle dependent elements, one cell cycle homology region and numerous Sp1 sites. PCR-based analysis of human genomic DNA, digested with methylation-sensitive and -insensitive restriction enzymes, indicated that the CpG island was unmethylated in both normal and neoplastic tissues. Primer extension and S1 nuclease mapping of the human survivin gene identified two main transcription start sites at position -72 and within -57/-61 from the initiating ATG. Transfection of cervical carcinoma HeLa cells with truncated or nested survivin promoter-luciferase constructs revealed the presence of both enhancer and repressor sequences and identified a minimal promoter region within the proximal -230 nt of the human survivin gene. Unbiased mutagenesis analysis of the human survivin promoter revealed that targeting the Sp1 sequences at position -171 and -151 abolished basal transcriptional activity by approximately 63-82%. Electrophoretic mobility-shift assay with DNA oligonucleotides confirmed formation of a DNA-protein complex between the survivin Sp1 sequences and HeLa cell extracts in a reaction abolished by mutagenesis of the survivin Sp1 sites. These findings identify the basal transcriptional requirements of survivin gene expression.